1. Date of download: 5/28/2016 From: Changes in DNA Methylation in Neoplasia: Pathophysiology and Therapeutic Implications Ann Intern Med. 2001;134(7):573-586. doi:10.7326/0003-4819-134-7-200104030-00011 Structure of cytosine, 5-methylcytosine, and hypomethylating 5-methylcytidine analogues. Figure Legend: Copyright © American College of Physicians. All rights reserved.American College of Physicians

2. Date of download: 5/28/2016 From: Changes in DNA Methylation in Neoplasia: Pathophysiology and Therapeutic Implications Ann Intern Med. 2001;134(7):573-586. doi:10.7326/0003-4819-134-7-200104030-00011 The maintenance methylation process.Top.3Middle.3MtaseBottom.left5-AzarightSome (but not all) cytosine-followed-by-guanosine (CpG) sites carry a methyl (CH ) group at the 5′ position of cytosine. Both strands of DNA are methylated (“fully methylated”). Immediately after DNA replication, the newly formed strand is unmethylated (no CH groups), resulting in hemimethylated double-stranded DNA, which rapidly attracts one of several methyltransferase ( ) enzymes. Through the action of methyltransferase enzymes, methyl groups are added symmetrically to hemimethylated CpG sites ( ), resulting in fully methylated DNA. In the presence of cytidine analogues ( ), methyltransferases are depleted, and the process of remethylation after replication is inhibited, leading to DNA that remains hemimethylated ( ). With subsequent rounds of replication in the absence of methyltransferases, DNA becomes progressively more demethylated, until most DNA molecules are completely unmethylated. Figure Legend: Copyright © American College of Physicians. All rights reserved.American College of Physicians

3. Date of download: 5/28/2016 From: Changes in DNA Methylation in Neoplasia: Pathophysiology and Therapeutic Implications Ann Intern Med. 2001;134(7):573-586. doi:10.7326/0003-4819-134-7-200104030-00011 Effects of methylation and histone deacetylation on gene expression and silencing.topblack boxesovalsarrows(leftmMBPbottomHDACrighttopInitially ( ), genes ( ) are unmethylated, and the promoter region is occupied by transcription factors ( ) that direct production of messenger RNA ( ). De novo methylation, by itself, has a minimal effect on gene expression ). However, methylated DNA ( ) attracts methyl-binding proteins ( ), such as MeCp2 ( ). These methyl-binding proteins in turn attract a protein complex that contains histone deacetylases ( ). At this point, synthesis of messenger RNA synthesis is inhibited and no functional protein can be made from the gene. Through the action of methyl-binding proteins and histone deacetylases, the DNA structure changes to a compact, “condensed chromatin” configuration ( ), which results in permanent inhibition of messenger RNA and protein production (silencing). Hypomethylating agents can reverse this silenced state and restore messenger RNA and protein expression ( ). Histone deacetylase inhibitors act synergistically with hypomethylating agents to restore functional gene expression. Figure Legend: Copyright © American College of Physicians. All rights reserved.American College of Physicians